Mg chemistry is unique among biological cations, and cells possess novel mechanisms for regulating Mg2+ and facilitating its passage through membranes. This proposal focuses on the CorA Mg2+ transporter, the primary Mg2+ uptake system for most Eubacteria and Archaea. The crystal structure of CorA from Thermotoga maritime has been determined to 3.9 A resolution. It is a funnel-shaped homopentamer with two transmembrane (TM)helices. The extracellular region is composed of only a short conserved loop that connects the two TM helices. The channel is composed of five helices, one from each monomer and appears gated by the side chains of bulky hydrophobic residues within the pore. These helices extend well beyond the membrane through the cytoplasmic domain, forming the funnel inner surface. Outside the funnel, the cytoplasmic neck of the pore is surrounded by a ring of highly conserved positively charged residues. Two negatively charged helices in the cytoplasmic domain extend back towards the membrane outside the funnel and abut the ring of positive charge. These exterior helices may serve to counteract the positively charged ring, suggesting a gating mechanism. An apparent Mg2+ ion was bound in the cytoplasmic domain between monomers, linking the extended helix from one monomer to a set of helices in another. The Mg2+ binding site, conserved in CorA orthologs, may link pore opening to the intracellular concentration of Mg2+. Aim 1 will continue study of CorA and its mechanism of Mg2+ transport using site directed mutagenesis and transport assays to probe the hypothesized mechanism of transport and gating. Aim2 will continue structural work on the T. maritima CorA including determination of an open pore form of CorA and to determine the structure of the soluble domain of S. Typhimurium CorA. Aim 3 will focus on other members of the large CorA family. Bacterial ZntB has modest sequence identity to CorA but mediates efflux of Zn2+ rather than influx of Mg2+. Eukaryotic Mrs2 proteins mediate Mg2+ influx into mitochondria but have sequence similarity with CorA only in the membrane domain. We propose to test the hypothesis that the structures of ZntB and Mrs2 are identical to that of the CorA Mg2+ transporter by determining the crystal structures of the soluble domains of both transporters. Health relevance. Study of CorA is important because it is a virulence factor in prokaryotes and thus an antibiotic target, In addition, it mediate Mg2+ flux into mitochondria and thus is important in control of Mg2+ homeostasis in eukaryotic cells and possibly in the mitochondrion's role in apoptosis.